Peroxiredoxins are thiol-specific antioxidant proteins that protect cells from ROS-induced damage by reducing cellular peroxides. They have been implicated in numerous cellular processes affected by ROS including cell signaling, proliferation, and apoptosis, as well as diseases related to oxidative stress such as atherosclerosis and aging. Several recent studies have reported overexpression of peroxiredoxins in cancer. However, certain members of this family have now been implicated as tumor suppressor proteins. Prdx1 and Prdx6 are two structurally and functionally distinct peroxiredoxins that are overexpressed in breast cancer. Tumor suppressor activity has now been demonstrated for Prdx1, and suggested for Prdx6. We have found that the expression of these genes is significantly higher in the breast adenocarcinoma cell line MCF7, as compared to the non- cancerous MCF10A breast epithelial line, and expression is associated with the proliferative state of the cell. We have found that PKC and MAPK can regulate these genes, and have identified putative transcription factor binding sites in the Prdx6 promoter that may mediate expression. The broad, long- term objectives of this project are to understand the function of Prdx1 and Prdx6 in cancer cells, and determine the mechanism by which they are overexpressed. We hypothesize that these proteins will play distinct roles in normal vs. cancer cells. Three specific aims are proposed to address these objectives. The first aim will examine the effect of Prdx1 and Prdx6 suppression in the MCF10A and MCF7 cell lines. This aim will be carried out using transient transfections with small interfering RNA (siRNA) for mRNA-targeted degradation of Prdx1 and Prdx6, and subsequent measurement of cellular peroxides, oxidative DNA damage, and apoptosis. The second aim will compare the function of Prdx1 and Prdx6 proteins in these two cell lines. We will determine Prdx1 and Prdx6 peroxidase activity, interactions with known molecular partners, and their ability to regulate signal transduction and transcription factor activity. The third aim will examine whether ROS-induced signaling pathways are responsible for Prdx1 and Prdx6 upregulation in MCF7 cells. We will measure peroxiredoxin expression in MCF7 cells treated with inhibitors of ROS, PKC and MAPK. Finally, we will test nuclear extracts from the two cell lines for binding activity of candidate regulatory transcription factors (Sp1, AP1, Egr1, SRF, E2F, c-myc, p53, Nrf1 and Nrf2) and binding to putative binding sites in the Prdx1 and Prdx6 promoters. With the recently emerging data implicating these genes in cancer, the proposed studies will provide essential insight into their mechanism of action and regulation. PUBLIC HEALTH RELEVANCE: Relevance The proposed series of experiments will focus on the role of human peroxiredoxin genes in breast cancer. These genes encode antioxidant proteins that protect cells from harmful oxidative damage, and they are overexpressed in many human cancers. Since oxidative damage can lead to both DNA mutation and cell death, these proteins are likely to play an important and complex role in cancer in humans.